Authors’ Reply: Serum Protein-induced Tubular Injury
Authors: Kevin Lidberg, Jonathan Himmelfarb, Edward Kelly, Shreeram Akilesh
Journal: Journal of the American Society of Nephrology (2022)

We thank Dr. O'Neill for his interest in our recent study,1 which showed that serum components other than albumin drive a proinflammatory response in proximal tubule epithelial cells (PTECs) in vitro—a finding that was supported by observations in a preclinical model of renal fibrosis and in patients with proteinuric kidney disease. In his letter, Dr. O'Neill points out an important difference in the composition of serum versus plasma—namely, that clotting of blood to generate serum can increase the levels of certain soluble factors due to platelet activation such as chemokines, cytokines, and growth factors, which may have a confounding effect on our interpretation of a proinflammatory tubular response to serum.2 However, the use of plasma in cell culture, and especially microfluidic cell culture, presents notable challenges and drawbacks as outlined below. First, plasma contains high levels of fibrinogen, which can be activated to form insoluble fibrin polymers. To prevent fibrin formation, plasma must be supplemented with an anticoagulant such as heparin, ethylenediaminetetraacetic acid (EDTA), or citrate. As EDTA and citrate are calcium chelators, they are ineffective at preventing fibrin formation when plasma is diluted in calcium containing cell culture media. Furthermore, fibrin formation in a microfluidic device could occlude media flow and prevent treatments from reaching the cells or lead to cell injury. Heparin, on the other hand, is a polyanionic glycosaminoglycan and is closely related to endogenous heparan sulfate. Heparan sulfate is a ligand for Toll-like receptor 4 (TLR4), a receptor expressed by kidney tubular epithelial cells and whose activation is associated with a proinflammatory response in mice.3,4 Therefore, EDTA and citrated plasma are likely to occlude the microfluidic device, whereas heparin-treated plasma may cause an inflammatory PTEC response due to TLR activation. A potential solution is to use recalcified plasma in which fibrinogen is removed from plasma by centrifugation after calcium supplementation. We recognize the potential confounding effect that platelet-derived factors could have on the PTEC response to serum in our study and plan to compare proximal tubule cell response to serum and recalcified plasma directly in the future. Dr. O'Neill also points out that the serum used in this study was not allogeneic and could contain autoantibodies. Plasma suffers from this same limitation because it contains antibodies and is also not allogeneic. Even if the serum contained autoantibodies, it is not certain that this would significantly affect PTEC phenotype without effector immune cells. In support of this idea, we have previously treated PTECs with IgGs purified from human serum and did not observe a change in excretion of KIM-1. Finally, Dr. O'Neill questioned whether it can be concluded from our study that nonselective proteinuria contributes to tubular injury. We show that serum, but not albumin, treated PTECs activated canonical stress and injury-associated genes and increased production and secretion of injury and inflammation-associated proteins. These phenotypic changes were mirrored in multiple preclinical models of tubular injury and renal fibrosis. Furthermore, similar findings were seen in tubulointerstitium gene expression data in biopsies from patients with minimal change disease and focal and segmental glomerulosclerosis in the NEPTUNE cohort. This weight of evidence supports our original conclusion that serum factors other than albumin can contribute to a proinflammatory response in PTECs and lead to progressive kidney injury. Disclosures S. Akilesh reports research funding from GoldfinchBio, Inc.; honoraria from invited seminars; and participation in a speakers' bureau for NanoString. J. Himmelfarb reports consultancy for Akebia Therapeutics, Chinook Therapeutics, Maze Therapeutics, Pfizer, RenalytixAI, and Seattle Genetics; ownership interest in Kuleana Technology, Inc.; honoraria from various academic institutions for invited lectures; patents held by the University of Washington; an advisory or leadership role for CJASN (editorial board), BMC Medicine (editorial board), and Nature Reviews Nephrology (advisory board); and other interests or relationships with Northwest Kidney Centers (research grant support) and Kuleana Technology, Inc. (founder, CEO, president, and equity holder). E.J. Kelly reports working as a consultant for Nortis, Inc. K.A. Lidberg reports being an employer of RayzeBio. Funding None.